高糖诱导大鼠血管平滑肌细胞转分化的作用

High glucose induces vascular smooth muscle cells phenotype transformation

目的:观察高糖对体外培养的大鼠胸主动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)功能以及与成骨细胞转分化相关蛋白表达的变化,探讨糖尿病血管病变的可能细胞/分子发病机制。方法:用组织贴块法建立SD大鼠胸主动脉VSMCs的体外培养技术;高糖(葡萄糖浓度:50 mmol/L)作用该细胞,设相同浓度甘露醇为对照组,观察在不同的培养时间下(3、12h和第3、6、9、12d),采用免疫蛋白印迹法和间接免疫荧光法分别检测α平滑肌肌动蛋白(α-SMA),骨桥蛋白(osteopontin,OPN)和成骨细胞特异性核转录因子(core-binging factorα-1,Cbfα-1/RUNX 2)的表达。细胞钙沉积含量测定观察培养细胞钙盐的沉积。结果:组织贴块法培养的大鼠原代胸主动脉VSMCs呈梭型,α-SMA表达强阳性;高糖作用12h,VSMCs的Cbfα-1表达明显上升;高糖作用9d后,VSMCs的α-SMA表达量较高糖作用前降低近90%;免疫荧光染色显示VSMC细胞呈低强度的α-SMA染色阳性;同时,高糖作用下的VSMCs能够检测到OPN蛋白的表达,与对照组相比差异显著;高糖作用下细胞钙沉积含量明显增加。结论:持续的高糖作用能够使动脉VSMCs发生向类似成骨样细胞的转分化,提示高糖诱导的VSMCs转分化可能参与糖尿病血管病变的发生发展。

Objective：To investigate the effects of high glucose on vascular smooth muscle cells（VSMCs） cultured in vitro and proteins expression associated with the phenotype transformation. Methodology：Primary culture of VSMCs was carried out using an explant method.VSMCs were incubated with high glucose（50mmol/L） medium for different times.The expressions of core binding factorα1（Cbf α-1）,osteopontin（OPN） and α-smooth muscle actin（α-SMA） were measured by Western blot and immunofluorescence staining.The cellular mineral deposition was assessed by testing the concentration of calcium. Results：（1）VSMCs was isolated from thoracic aortic tissue by explant method,and the expression of α-SMA was positive;（2）Time-course studies revealed that up-expression of osteoblast-specific transcription factor Cbfα-1 was occured as early as 12 hours.Western blotting exhibited VSMCs lost their lineage marker α-SMA by proximally 90% after incubated with high glucose medium for 9 days compared with the control.The immunofluorescence staining of VSMCs incubated with high glucose medium for 9 days was weak.OPN was up-regulated significantly when VSMCs were incubated in high glucose medium.（3）The granular calcium deposition was increased in VSMCs with high glucose. Conclusion：Continuous high glucose incubation can induce osteoblast phenotype transformation of VSMCs.It suggested that VSMCs phenotype transformation induced by high glucose could contribute to the development of diabetic vascular disease.